The Drosophila tumor necrosis factor Eiger promotes Myc supercompetition independent of canonical Jun N-terminal kinase signaling

Abstract Numerous factors have been implicated in the cell–cell interactions that lead to elimination of cells via cell competition, a context-dependent process of cell selection in somatic tissues that is based on comparisons of cellular fitness. Here, we use a series of genetic tests in Drosophila to explore the relative contribution of the pleiotropic cytokine tumor necrosis factor α (TNFα) in Myc-mediated cell competition (also known as Myc supercompetition or Myc cell competition). We find that the sole Drosophila TNF, Eiger (Egr), its receptor Grindelwald (Grnd/TNF receptor), and the adaptor proteins Traf4 and Traf6 are required to eliminate wild-type “loser” cells during Myc cell competition. Although typically the interaction between Egr and Grnd leads to cell death by activating the intracellular Jun N-terminal kinase (JNK) stress signaling pathway, our experiments reveal that many components of canonical JNK signaling are dispensable for cell death in Myc cell competition, including the JNKKK Tak1, the JNKK Hemipterous and the JNK Basket. Our results suggest that Egr/Grnd signaling participates in Myc cell competition but functions in a role that is largely independent of the JNK signaling pathway.

The studies instead suggest that Dredd signaling plays a role in competition-induced apoptosis.
The most interesting and novel part of the paper is the suggestion JNK activation is not directly connected to competitive status and that instead cellular elimination relies on a combination of JNK and TLR signaling components.Unfortunately, the data in this paper only suggests this possibility rather than clearly demonstrating and elucidating the CCSM.
My main concern about claiming true JNK independence and a reliance instead on Dredd is the reliance on studies using overexpression of Dredd and an RNAi knockdown of Grindelwald Dredd overexpression studies.Figure 5.
• The authors assert that overexpression of Dredd enhances "loser" cell elimination.This is not obvious from the data and there appears to be no direct comparison of WT clone area compared to +Dredd clone areas.The reduced clone area of the +Dredd controls suggests an overall diminished area which would make determining an enhanced elimination more difficult.Statistical analysis across WT and +Dredd or between WT control v losers and +Dredd control v losers would be necessary to provide evidence for this.
• The lack of significance between the grndIR control clone area size and the losers (Figure 5F) is used to argue competition is Grnd-dependent.However, the lack of significance appears to rely on a few outliers in each category.There also appear to be a disproportionate number of experimental repeats in these categories, which leads to a questioning of how robust this lack of difference is.
• When combining overexpression of Dredd and an RNAi knockdown of Grindelwald, there appears to be some complicating factors at play.Combined overexpression and knockdown appear to lead to reduced control clone area compared to Dredd+ alone control clone area.This is interesting given that GrndIR alone does not lead to any reduction in control clone area.While statistically this may not be the case, it would be important to run these tests if using the control as a standard against which to compare elimination.
• The statistical difference between control and loser clone size in Dredd+GrndIR also seems to have required many more tests to establish than any other of the comparisons.I am concerned that experiments were repeated until the statistics worked out rather than there still being robust elimination due to cellular competition.• There is a lack of a Data Availability statement that would allow readers to dive deeper into this.
Even beyond the concerns with Figure 5, the limitations of this model should be considered.Super-competition is just one of the types of cellular competition being studied.There is significant evidence this competition and its signaling pathways may be distinct from Xrp1-dependent or other forms of cellular elimination.It should also be noted that studies have shown an interplay between Myc and JNK.The reliance of a system where even modest overexpression of Myc is what is used to drive winner status could influence JNK signaling pathways.
Without a clear link between Grindelwald/Traf6/Traf8 activation and Dredd signaling, the paper suggests but does not supply strong evidence for their proposed CCSM.The work is interesting and, with the exception of Figure 5, is good.However, their main assertion of a but not as convincing.
My other comments are more minor.
• A lack of a direct correlation between JNK transcriptional activity and "winner" vs "loser" status (especially given that there is some low-level activity in all cells) does not clearly eliminate the possibility that JNK activity is still central to cellular elimination due to competition.(Figure 1F-G).
• The importance and relevance of ectopic expression of efr in Figures 4B-C is unclear.This introduces a new approach whose direct comparison and relationship to the other studies looking at clonal populations in the wind disk is unclear.
• There is no mention of the grndIR flies in the methods.
• It would be helpful to explicitly state that a constitutively active form of the intracellular domain of Grnd is being used rather than just an intracelluar domain.
• Elimination is misspelled at the bottom of page 9.
Reviewer #2 (Comments for the Authors (Required)): Critique of Kodra et al Kodra and colleagues present new and important finding of the mechanism of Myc super-competition.Super-competition is the process by which cells with higher fitness due to increases in genetic dose of Myc eliminate wild-type (WT) neighboring cells.The process of cell competition and super-competition play important roles in a wide array of processes (from embryonic development to tumor progression) but there are critical gaps in our knowledge about the molecules involved.Previously the Johnston lab has identified what they term the cell competition signaling module (CCSM) comprising some components of Toll and IMD pathways in the elimination of losers.These are: several Toll related receptors (TRRs), the secreted Toll ligand Spatzle (Spz), and 2 spz-cleaving proteases, the adaptor FADD, the caspase-8 homolog Dredd, and the NF-kB factor, Relish (Rel).The role of the JNK pathway in Myc super-competition is not well understood, and the authors set out to test its function using a set of elegant genetic experiments.The authors find (1) the TNF-like factor Eiger (Egr) is required in WT losers for their outcompetition; (2) The Egr receptor Grnd is also required in WT losers for their elimination but the other TNFR in flies -Wgn -is not; (3) expression of the Grnd intracellular domain is sufficient to induce death in non-competitive and competitive scenarios; (4) Traf4 and Traf6 are required in losers for their elimination; (5) surprisingly, the Tak1 kinase that acts downstream of Egr/Grnd does not regulate loser death in competitive scenarios, and reduction of JNKK (Bsk) and JNK (Hep) cannot suppress the death of losers.These data suggest that Egr/Grnd function in losers is independent of JNK activity, and indeed the authors provide data to support this model in the last figure of the paper.Taken together these results support a model in which activation of Grnd by Egr during cell competition leads to activation of the FADD/Dredd/Rel and then apoptosis (hid) and in parallel to the activation of JNK signaling, which modestly contributes to loser death.Overall, the data are of high quality, the assays are robust, and the genetics are very solid.These results advance the field of cell competition and would be of interest to a wide range of geneticists.I have some suggestions for the authors.1.Some of the references are properly formatted but others lack the publication year or still have the brackets of the bibliographic software (Endnote).2. I suggest that the authors move the model from Suppl Fig 1 to main body Fig. 1.I also suggest that they add the Spz proteases, etc so that it matched the text.When the authors write about Egr/Grnd activating the CCSM, it is not clear to me whether this proceeds from Egr/Grnd to Spz/Tl to FADD/Dredd/Rel/Hid or whether Spz/Tl is not involved.In other words, I am asking for a bit more precision about how Egr/Grnd activates CCSM.I think at some clarity in the model would be helpful to the readers.3. The figures are pixelated so I suggest uploaded higher resolution images.4. The authors need to provide statistically analysis of Fig. 5D vs. Fig.5B since they use the word "significantly" but do not provide a P value.
Reviewer #3 (Comments for the Authors (Required)): The manuscript of Laura Johnston and colleagues addresses the role of the TNF/JNK signalling pathway in dMyc driven cell competition using the wing primordium of Drosophila as model system.Authors conclude that Eiger (not coming from the wing epithelium, although this should be tested by RNAi), its receptor Grindelwald (but not Wengen), Traf4 and Traf6 (but not Hep and JNK) are required in loser cells to undergrow and get outcompeted.Paper is well written, figures self-explanatory and this topic is timely for the Genetics journal.I have the following comments, though, that should be raised by the authors (1) Intro is, to a large extent, devoted to summarize previous results from the field including the publications of their own lab on the CCSM.I would suggest to make the Intro shorter and to the point of the potential role of Eiger/JNK axis in cell competition.Otherwise, the reader might get unfocused.
(2) The proposal that Eiger is not functioning in a paracrine manner should be reinforced by functional data (RNAi for eiger?) (3) The no-role of Tak1 and Tak2 should be reinforced by the use of RNAi forms of these two genes.The fact that mutant larvae are viable either indicates that mutants are not completely null or there is strong maternal contribution.(4) I am not fully convinced about the conclusion that Hep and JNK have no role.There is a clear partial rescue of the size of loser clones!! Again, the fact that the hep allele is viable indicates that this mutant is not completely null or there is strong maternal contribution.The use of RNAi forms of heo and bsk should be able to reinforce the data (5) I am no convinced by the data on the potential connection Dredd-Grindelwald present in Figure 5.I think it should be removed.(6) References are not ok.

Associate Editor Comments:
I think that the manuscript could be improved by editing for clarity in some places.Here are a few suggestions... pg 3 "Reporters of the JNK stress pathway such as puc-lacZ, an enhancer trap insertion in the puckered (puc) locus (encoding a phosphatase antagonist of JNK activity) (MARTINBLANCO et al. 1998;MCEWEN AND PEIFER 2005) indicate that JNK activity is present in some epithelial cells of the wing and eye imaginal discs during their normal growth, where it promotes cell survival by blocking JNK activity (MCEWEN AND PEIFER 2005;WILLSEY et al.)."I am guessing that the "it" in the last clauses refers to puc in the first clause.pg 9 "However, in the egr3AG background, the egr mutant clones generated in both competitive and non-competitive contexts grew at the same rate and to a similar final size" I think that calling these egr mutant clones is a bit confusing.They are egr mutant, but so is the rest of the tissue.pg 13 "If so, we reasoned that in this competitive context, activation of Egr/Grnd signaling in a Tak1 mutant should not suppress loser cell death (i.e., the cells would still be eliminated)."I suggest rewording to "...a Tak1 mutant should not suppress Egr/Grnd induced cell death."I am pleased to accept your manuscript entitled "The Drosophila TNF Eiger promotes Myc super-competition independent of canonical JNK signaling" for publication in GENETICS, pending minor revision.I think that you did a great job revising the manuscript in response to reviewers concerns.
One of the last things to fix are the figure 2 panel labels which need to be reordered to match text and figure legend (i.e.b, b' vs c, c', it happens).
One minor suggestion is that you could be even more explicit on first definition of "winner" and "loser" clones so readers remember.The fact that the genotype of winner cells are 3x Myc and losers are 2x Myc is only mentioned once after that in both text and figure legends.You may also wish to remind the reader in select places.I also wouldn't choose to call those transformed flies wild type (WT), although I understand that you mean not mutant for other genes.
I am sorry to hear about your continued Endnote problems.I will leave it to the editorial office to advise how to proceed.Hopefully copy editors can help to fix the references in text.
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Fig 1 :
Fig 1: The order of the legend does not match figure panels.

Fig
Fig 3E,F -I find it a little confusing that the insets to these panels are different discs and time points.I suggest making them separate panels with time after induction noted.Fig 4I-J -There is a legend for these panels, but no panels in the figure.Include a data availability statement.Please update FlyBase ref to: https://academic.oup.com/genetics/article/227/1/iyad211/7596147?login=true FYI: Genetics has switched to lower case letters for figure panels.